Our long-term goal is to induce immunity and long-term immunological memory against autologous tumor in the tumor-bearing host. CD8+ T cells can be effective agents in tumor rejection, however, they usually require "help," from specifically activated CD4+ Th cells. In many cases immunity is not effective because CD4+ Th cells are not activated to tumor antigen. We have therefore hypothesized that tumor immune responses could be significantly improved if stimulation of tumor-specific CD4+ Th cells was more effective. Our strategy to improve presentation of tumor antigen has focussed on genetically modifying tumor cells so that they directly present tumor peptides to CD4+ Th cells, bypassing the need for professional antigen presenting cells (APC). During the first 4.5 years of this grant, we have used gene transfer to express syngeneic MHC class II genes in sarcoma and melanoma cells such that the genetically modified tumor cells directly present class II/tumor peptide to the responding CD4+ T cells. These transfectants are very effective immunogens for inducing long-term, specific immunity against wild type tumor. Tumor cell expression of the costimulatory molecule B7 along with syngeneic MHC class II yields cells that are potent immunotherapeutic agents for the treatment of established sarcomas. During the next 5 years, our goals are two-fold: l) Develop the future therapeutic use of these transfectants for treatment of established tumor and the prevention of metastatic disease. 2) Ascertain the mechanism by which th transfectants induce immunity. These goals will be accomplished.through the following specific aims: l) Enhance the ability of class II transfected tumor to activate CD4+ T cells by transfecting them with genes encoding molecules specific to professional APC (B7-2, CD48, heat stable antigen, ICAM-1), and genes encoding cytokines that stimulate CD4+ T cell differentiation (IL-12, IL- 1beta). 2) Determine if the transfectants can "rescue" mice carrying established wild type tumor. 3) Determine if the transfectants can be immunizing and/or immunotherapeutic agents for the prevention and/or treatment of metastatic disease. Determine if recurrence of primary tumor can be blocked by immunization with transfectants. 4) Ascertain how the transfectants stimulate tumor-specific immunity. We assume that if we understand the mechanism(s) by which the transfectants stimulate the immune response, we will be better able to manipulate them as therapeutic agents. Completion of these studies will therefore provide a strong foundation for future translational research employing this novel immunotherapeutic strategy.